U.S. patent number 4,569,790 [Application Number 06/594,223] was granted by the patent office on 1986-02-11 for process for recovering microbially produced interleukin-2 and purified recombinant interleukin-2 compositions.
This patent grant is currently assigned to Cetus Corporation. Invention is credited to Wolf Hanisch, Kirston Koths, Michael Kunitani, James Thomson, Kenneth Wilson.
United States Patent |
4,569,790 |
Koths , et al. |
February 11, 1986 |
**Please see images for:
( Certificate of Correction ) ** |
Process for recovering microbially produced interleukin-2 and
purified recombinant interleukin-2 compositions
Abstract
A process for recovering microbially produced IL-2 in a highly
pure form from the cellular material of the microorganisms that
produced it comprising: disrupting the cell membranes of the
microorganisms; extracting the disruptate with a chaotropic agent,
such as urea, that selectively extracts microbial proteins from the
cellular material; solubilizing the IL-2 in the solid phase of the
extraction mixture with an aqueous solution of a solubilizing
agent, such as SDS, containing a reducing agent; and separating the
IL-2 from the resulting solution by an optional extraction with
2-butanol or 2-methyl-2-butanol followed by gel filtration
chromatography, oxidizing the IL-2 and purifying the oxidized IL-2
by RP-HPLC.
Inventors: |
Koths; Kirston (Berkeley,
CA), Thomson; James (Albany, CA), Kunitani; Michael
(Oakland, CA), Wilson; Kenneth (Walnut Creek, CA),
Hanisch; Wolf (Oakland, CA) |
Assignee: |
Cetus Corporation (Emeryville,
CA)
|
Family
ID: |
24378044 |
Appl.
No.: |
06/594,223 |
Filed: |
March 28, 1984 |
Current U.S.
Class: |
530/351;
435/69.52 |
Current CPC
Class: |
A61P
35/00 (20180101); C07K 14/55 (20130101); C07K
1/1133 (20130101); A61K 38/00 (20130101) |
Current International
Class: |
C07K
1/00 (20060101); C07K 1/113 (20060101); C07K
14/435 (20060101); C07K 14/55 (20060101); C07K
003/12 (); C12D 021/02 () |
Field of
Search: |
;260/112R,112.5R
;435/170,172,849,68 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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83103582.9 |
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Oct 1983 |
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EP |
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92163 |
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Oct 1983 |
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EP |
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83400938.3 |
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Nov 1983 |
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EP |
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94317 |
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Nov 1983 |
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EP |
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118977 |
|
Sep 1984 |
|
EP |
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119621 |
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Sep 1984 |
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EP |
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0121352 |
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Oct 1984 |
|
EP |
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0128467 |
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Dec 1984 |
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EP |
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147819 |
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Jul 1985 |
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EP |
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0148098 |
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Nov 1980 |
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JP |
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Other References
Watson et al., J. Exp. Med., (1979), 150:849-861. .
Gillis et al., "J. Immunol., (1980), 124:1954-1962. .
Mochizuki et al., J. Immuno. Meth., (1980), 39:185-201. .
Welte K. et al., J. Exp. Med., (1982), 156:454-464. .
Derynel R. et al., Nature, (1980), 287, 193-197. .
Zaniguchi et al., Nature, (1983), 302, p. 305, Structure and
Expression of a Cloned . . . IL-2. .
Lymphotine Res. 1(2) 1982, FASEB 1982 Editorial. .
J. Immunol. 129(6), 1982, Ihle et al., p. 2431, Procedures for the
Purification of IL--3 to Homogeniety. .
Milestone et al., Purification of T-Cell Growth Factor to
Homogeneity by RP-HPLC, Biochem. Biophy. Res. Comm. 115(3) 1983,
pp. 762-768 (1983). .
Henriksen et al., Control of the Expression of IL-2 Activity, Cell.
Immunol. 73 (1982), pp. 106-114. .
Henderson et al., A Rapid, Large Scale Purification Procedure for
Gibbon IL-2, J. Immunol. 131(2) (1983), pp. 810-815. .
Riendeau et al., Purification of Mouse IL-2 to Apparent
Homogeneity, J. B. C. 258(20) 1983, pp. 12114-12117. .
Devos et al., Nucleic Acid Res. 11(13), 1983, p. 4307, Molecular
Cloning of Human IL-2-CDNA . . . E Coli. .
A. S. Stern et al., "Purification . . . ", Proc. Natl. Acad. Sci.
(USA), 81, 871-875, (1984). .
R. J. Robb, "Interleukin 2: The Molecule and its Function",
Immunology Today, vol. 5 (7), (1984). .
Coughlin, R. T., et al., Federation Proceedings, 42(7), 2022 (May
1, 1983), "Reverse Phase High Performance Liquid Chromatography of
Lipopolysaccharides". .
Bio/Technology, Dec. 1984, pp. 1035-1038, "Chromatographic Removal
of Pyrogens"..
|
Primary Examiner: Kight; John
Assistant Examiner: Draper; Garnette D.
Attorney, Agent or Firm: Halluin; Albert P. Ciotti;
Thomas
Claims
We claim:
1. A process for recovering purified, oxidized human IL-2 from a
transformed microorganism containing the IL-2 comprising:
(a) disrupting the cell membrane of the microorganism;
(b) extracting the disruptate with an aqueous solution of a
chaotropic agent that extracts non IL-2 proteins selectively from
the cellular material and from the IL-2;
(c) solubilizing the IL-2 in the solid phase of the extraction
mixture with an aqueous solution of a solubilizing agent that forms
a water soluble complex with the IL-2, said solution containing a
reducing agent to thereby fully reduce the IL-2 in the complex;
(d) separating the reduced IL-2 from the resulting solution in the
presence of the reducing agent;
(e) oxidizing the reduced IL-2;
(f) further purifying the resulting oxidized IL-2 product by gel
filtration or reverse-phase high performance liquid chromatography
as individual or combined steps; and
(g) recovering a purified oxidized recombinant human IL-2
composition having an IL-2 content of at least about 95% as
determined by reducing SDS-PAGE analysis, an endotoxin content of
less than about 0.1 nanograms/mg of IL-2 and substantially free of
pyrogens as determined by the U.S.P. rabbit pyrogen test at a
dosage of 3.3.times.10.sup.5 U/kg.
2. The process of claim 1 wherein the chaotropic agent is urea.
3. The process of claim 2 wherein the concentration of urea in the
extraction mixture is in the range of about 3.5 M to 4.5 M.
4. The process of claim 1 wherein step (b) is carried out at a
basic pH.
5. The process of claim 4 wherein the pH is in the range of about
8.1 to about 8.5.
6. The process of claim 1 wherein the solubilizing agent is sodium
dodecyl sulfate or sodium lauryl sarcosine.
7. The process of claim 1 wherein step (f) is carried out by
reverse phase high performance liquid chromatography at a pH in the
range of 2.1 to 2.3 using a bonded phase wide pore silica gel and a
gradient solvent system comprising an organic acid and an organic
solvent for eluting IL-2.
8. The process of claim 7 wherein the solvent system is acetic
acid-propanol, trifluoroacetic acid-propanol, or trifluoroacetic
acid-acetonitrile.
9. The process of claim 1 wherein step (f) is carried out by
isolating an IL-2-containing fraction from the solution by gel
filtration and purifying the resulting IL-2 from the fraction by
reverse-phase high performance liquid chromatography.
10. The process of claim 9 wherein the reverse phase high
performance liquid chromatography is carried out at a pH in the
range of 2.1 to 2.3 using a bonded phase wide pore silica gel and a
gradient solvent system comprising an organic acid and an organic
solvent for IL-2.
11. The process of claim 10 wherein the solvent system is acetic
acid-propanol, trifluoroacetic acid-propanol, or trifluoroacetic
acid-acetonitrile.
12. The process of claim 1 including the following additional steps
of:
(i) extracting the IL-2 from the aqueous solution of (c) with
2-butanol or 2-methyl-2-butanol;
(ii) acid precipitating the IL-2 from the extract; and
(iii) purifying the acid precipitated IL-2 by gel filtration.
13. The process of claim 12 wherein the chaotropic agent is urea,
the concentration of urea in the extraction mixture is 3.5 M to 4.5
M, and the extraction of step (f) is carried out at a pH of 5 to
7.5.
14. The process of claim 12 including purifying the resulting
oxidized gel filtered product by reverse-phase high performance
liquid chromatography.
15. Purified recombinant human interleukin-2 (IL-2) composition
wherein the IL-2 is unglycosylated and in oxidized form, having an
IL-2 content of at least about 95% as determined by reducing
SDS-PAGE analysis, an endotoxin content of less than about 0.1
nanogram/mg of IL-2 and substantially free of pyrogens as
determined by the U.S.P. rabbit pyrogen test at a dosage of
3.3.times.10.sup.5 U/kg.
16. The purified recombinant IL-2 composition of claim 15, wherein
the IL-2 content is greater than about 98% as determined by
reducing SDS-PAGE.
17. The purified recombination human IL-2 composition of claim 15,
wherein the IL-2 content is greater than about 98% as determined by
RP-HPLC.
18. The purified recombinant human IL-2 of claim 15, wherein the
IL-2 is des-alanyl, serine.sub.125 IL-2.
19. The purified recombinant human IL-2 of claim 16, wherein the
IL-2 is des-alanyl, serine.sub.125 IL-2.
20. The purified recombinant human IL-2 of claim 17 wherein the
IL-2 is des-alanyl, serine.sub.125 IL-2.
21. The process of claim 7 wherein the solvent system is acetic
acid-propanol.
Description
TECHNICAL FIELD
This invention is in the field of biochemical engineering. More
particularly, the invention concerns a biochemical separation or
recovery process in which interleukin-2 (IL-2) is separated or
recovered from microorganisms that have been transformed to produce
IL-2.
BACKGROUND ART
Native human IL-2 is an antigen-nonspecific, genetically
unrestricted soluble factor produced by erythrocyte rosette
positive T cells stimulated with antigens, mitogens and
alloantigens. It is a protein with a reported molecular weight in
the approximate range of 13,000 to 17,000 daltons (S. Gillis and J.
Watson, J Exp Med (1980) 159:1709) and an isoelectric point in the
approximate range of pH 6-8.5. Human IL-2 has a number of in vitro
and in vivo effects including enhancing the proliferative responses
of human peripheral blood mononuclear cells or murine thymocytes,
enhancing the immune response in humans and in animals against
bacterial, parasitic, fungal, protozoan and viral infections, and
supporting the growth of continuous T cell lines.
IL-2 and IL-2 muteins in which the cysteine residue at amino acid
125 has been replaced with serine and/or the initial alanine has
been eliminated have been produced microbially through genetic
engineering techniques. Microbially produced IL-2 is not
glycosylated and is produced in a reduced-state by the
microorganisms. When purified and oxidized, these microbially
produced IL-2s exhibit activity comparable to native human
IL-2.
Procedures for purifying native IL-2 from T cells are described by
Watson, J., et al, J Exp Med (1979) 150;849-861; Gillis, S., et al,
J Immunology (1980) 124:1954-1962; Mochizuki, D. Y., et al, J Immun
Meth (1980) 39:185-201; Welte, K., et al, J Exp Med (1982)
156:454-464; and European patent applications No. 83103582.9
(published 26 Oct. 1983 under No. 92163) and No. 83400938.3
(published 16 Nov. 1983 under No. 94317). In general these
procedures involve precipitating proteins from culture supernatants
with ammonium sulfate followed by a chromatographic
fractionation.
Commonly owned copending U.S. patent application Ser. No. 353,360,
filed 1 Mar. 1982 and Derynck, R., et al, Nature (1980) 287:193-197
describe procedures for recovering IFN-.beta. from
IFN-.beta.-producing E. coli. The patent application describes a
procedure in which IFN-.beta. is extracted from cellular material
with 2-butanol or 2-methyl-2-butanol.
DISCLOSURE OF THE INVENTION
The invention is a process for recovering IL-2 from an
IL-2-producing microorganism comprising:
(a) disrupting the cell membrane of the microorganism;
(b) extracting the disruptate with an aqueous solution of a
chaotropic agent;
(c) solubilizing the IL-2 in the solid phase of the extraction
mixture with an aqueous solution of a solubilizing agent that forms
a water soluble complex with the IL-2, said solution containing a
reducing agent; and
(d) separating the IL-2 from the resulting solution in the presence
of a reducing agent.
In preferred embodiments of this process the chaotropic agent is
urea at a concentration of about 3.5 M to about 4.5 M in the
extraction mixture, the solubilizing agent is sodium dodecyl
sulfate (SDS) or sodium lauryl sarcosine (sarcosyl), the
solubilized IL-2 is further extracted with 2-butanol or
2-methyl-2-butanol and the final separation is carried out by gel
filtration, the resulting sized product is oxidized and the
oxidized product is purified by reverse-phase high performance
liquid chromatography (RP-HPLC).
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a flow diagram of two alternative embodiments of the
invention process in which gel filtration chromatography is used as
a final purification step. The embodiment designated Method 1A uses
SDS as a solubilizing agent; the embodiment designated Method 1B
uses sarcosyl as a solubilizing agent. The figure includes
densitometer scans of SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) analyses of the product at various steps in the
process.
FIG. 2 is an HPLC chromatogram and SDS-PAGE analysis of the product
of Example 3.
FIG. 3 is an HPLC chromatogram of the product of Example 9.
FIG. 4 is a flow diagram of a preferred procedure for processing
microbially produced IL-2.
MODES FOR CARRYING OUT THE INVENTION
As used herein the term "IL-2" denotes an unglycosylated protein
that is (a) produced by a microorganism that has been transformed
with a human interleukin-2 gene or a modification of the human
interleukin-2 gene that encodes a protein having: (a) an amino acid
sequence that is at least substantially identical to the amino acid
sequence of native human interleukin-2 and (b) has biological
activity that is common to native human interleukin-2. Substantial
identity of amino acid sequences means the sequences are identical
or differ by one or more amino acid alterations (deletions,
additions, substitutions) that do not cause an adverse functional
dissimilarity between the synthetic protein and native human
interleukin-2. Examples of such proteins are the IL-2s described in
European patent application No. 83101035.0 filed 3 Feb. 1983
(published 19 Oct. 1983 under publication No. 91539) and European
patent application No. 82307036.2 filed 22 Dec. 1982 (published 14
Sept. 1983 under No. 88195), the IL-2s described in commonly owned
U.S. Ser. No. 564,224, filed 20 Dec. 1983, now U.S. Pat. No.
4,518,584, issued May 21, 1985, which description is incorporated
herein by reference, and the IL-2s described in the examples of
this application.
As used herein the term "transformed microorganism" denotes a
microorganism that has been genetically engineered to produce a
protein that possesses native human interleukin-2 activity.
Examples of transformed microorganisms are described in said
European patent publications Nos. 88195 and 91539, said U.S. Pat.
No. 4,518,584 and the examples of this application. Bacteria are
preferred microorganisms for producing IL-2. Synthetic IL-2 may
also be made by suitably transformed yeast and mammalian cells. E.
coli is particularly preferred.
The transformed microorganisms are grown in a suitable growth
medium, typically to an optical density (OD) of at least about 30
at 680 nm, and preferably between about 20 and 40 at 680 nm. The
composition of the growth medium will depend upon the particular
microorganism involved. The medium is an aqueous medium containing
compounds that fulfill the nutritional requirements of the
microorganism. Growth media will typically contain assimilable
sources of carbon and nitrogen, energy sources, magnesium,
potassium and sodium ions, and optionally amino acids and purine
and pyrimidine bases. (See Review of Medical Biology, Lange Medical
Publications, 14th Ed pp 80-85 (1980).) In expression vectors
involving the trp promoter, the tryptophane concentration in the
medium is carefully controlled to become limiting at the time IL-2
expression is desired. Growth media for E. coli are well known in
the art.
After the cells are harvested from the culture, they may be
concentrated, if necessary, to about 20 to 150 mg/ml, preferably 80
to 100 mg/ml (OD 40 to 300, preferably 160 to 200 at 680 nm) by
filtration, centrifugation, or other conventional methods.
Following concentration the cell membranes of the microorganisms
are disrupted. The main purpose of disruption is to facilitate the
following extraction and solubilization steps. Conventional cell
disruption techniques such as homogenization, sonication, or
pressure cycling may be used in this step of the process. Preferred
methods are sonication or homogenization with a Manton-Gaulin
homogenizer. The end point of the disruption step may be monitored
by optical density, with the optical density of the suspension
typically decreasing about 65% to 85%. In any event, the disruption
should break substantially all of the cells so that substantially
no intact cells are carried through to the solubilization step.
Before the disruption, the pH of the liquid phase of the
concentrate is adjusted, if necessary, to a level that facilitates
removal of E. coli proteins in subsequent steps, while retaining
IL-2 protein as an insoluble complex in the celluar debris. The pH
may be so adjusted by adding suitable buffers. In most instances
pHs in the range of about 8 to about 8.5 will be used.
The steps in the recovery process subsequent to the disruption step
are primarily designed to separate the IL-2 from E. coli proteins
to a high level of purity (preferably at least about 95% and more
preferably at least about 98%) in good yields while maintaining the
IL-2 in a reduced state. Simultaneously, these purification
processes, in combination, also reduce pyrogenic substances in the
final product to a level believed to be acceptable for patenteral
administration to patients.
After the cells have been disrupted the particulate matter may be
separated from the liquid phase of the disruptate and resuspended
in an aqueous medium buffered to the optimal pH for the extraction.
The particulate matter may optionally be washed with buffer at this
stage to remove any water soluble E. coli proteins therein. In any
event, the protein concentration of the cell suspension subjected
to the extraction will usually be in the range of about 5 to about
60 mg/ml, preferably 20 to 40 mg/ml.
The extraction of E. coli proteins from the particulate cellular
material may be carried out concurrently with the disruption or
sequentially following the disruption. It is preferably carried out
as a separate step following the disruption. The extractant is an
aqueous solution of a chaotropic agent (i.e., a mild protein
denaturant that dissociates hydrogen bonds and affects the tertiary
structure of proteins). The extractant selectively removes the bulk
of the E. coli proteins from the cellular debris leaving at least a
substantial portion of the IL-2 associated (contained in or bound
to) with the cellular debris. The selectivity is facilitated by the
hydrophobicity of the IL-2 and the fact that it is in a reduced,
insoluble state at a pH near the isoelectric point of the protein.
In addition, a substantial portion of the IL-2 may be present in
vivo as inclusion bodies of significant mass, as has been the case
with other cloned proteins expressed at high levels in E. coli.
Examples of extractants are urea and guanidinium hydrochloride
(guanidinium hydrochloride should not be used when SDS is used as a
solubilizing agent). Urea is preferred. The concentration of the
chaotropic agent in the extraction mixture will depend upon the
particular agent that is used and the amount of cellular material
in the extraction mixture. In the case of urea, concentrations
(final) between about 3.5 M and 4.5 M, preferably about 4 M, will
be used in batch processes at 25.degree. C. If the extraction is
run on a continuous basis over longer time periods it may be
desirable to use lower concentrations. Temperatures in the range of
20.degree. C. to 25.degree. C. will normally be used in extraction,
with room temperature being used for convenience. Mixing will
typically be used to enhance contact between the solution and
particulate matter and thus decrease the time required to extract
non-IL-2 proteins from the cellular debris. Kinetic analysis of the
extraction process was performed on the supernatants using
SDS-PAGE, and the extraction was found to be essentially complete
by 15-30 min.
Following the extraction, the mixture is separated into solid and
liquid phases. The IL-2 in the solid phase is then selectively
solubilized by contacting the solid phase with a neutral, aqueous
buffer containing a reducing agent and a solubilizing agent.
Surface active agents (detergents) that have a suitable
hydrophobic-hydrophilic balance to solubilize the hydrophobic IL-2
may be used. Alkali metal sulfates containing 10 to 14 carbon atoms
and alkali metal alkyl sarcosinates are preferred solubilizing
agents, with SDS and sarcosyl being particularly preferred.
The amount of solubilizing agent used in the solubilization will
depend upon the particular agent. When SDS or sarcosyl are used,
the preferred ratio (w/w) of SDS/sarcosyl to solid phase protein is
about 0.5:1 to 1.4:1. The solubilizing medium also contains a
sufficient amount of reducing agent to prevent the solubilized IL-2
from undergoing oxidation to any significant degree. Protein
reducing agents such as dithiothreitol (DTT) and 2-mercaptoethanol
may be used. The concentration of reducing agent such as DTT in the
medium will usually range between about 5 to 20 mM. The
solubilization will typically be carried out at temperatures in the
range of 20.degree. C. to 25.degree. C. with mixing to facilitate
contact between the solid phase and the solubilizing medium. Higher
temperatures may solubilize unwanted E. coli proteins. The
solubilization is considered complete when the sample has sat 15
min or the solution turns translucent. Insoluble material is
separated after completing the solubilization.
After the IL-2 is solubilized the IL-2 may optionally be extracted
from the aqueous solution under reducing conditions with 2-butanol
or 2-methyl-2-butanol to remove additional E. coli proteins,
notably including certain contaminants that have molecular weights
very close to the IL-2. Conditions (e.g., ionic strengths in the
range of 0.05 and 0.15) at which the aqueous solution and butanol
are substantially immiscible are used. In carrying out the organic
extraction the protein concentration of the aqueous solution is
preferably adjusted, if necessary, to less than about 6 mg/ml,
preferably about 0.5 to 4 mg/ml. Reducing conditions are maintained
by carrying out the extraction in the presence of a reducing agent
(e.g., DTT). The butanol will normally be added to the aqueous
solution of solubilized IL-2 in volume ratios in the range of about
1:1 to about 3:1 (extractant:aqueous solution), preferably about
1:1. The extraction may be carried out in a batch or continuous
operation. The temperature will normally be in the range of
20.degree. C. to 100.degree. C. and the pH will normally be about 4
to 9, preferably about 5 to 6. The time of contact between the
solution and the butanol is not critical and relatively short times
on the order of a few minutes may be used. After the extraction is
complete, the aqueous phase and butanol phase are separated and the
IL-2 is separated from the butanol phase. A preferred procedure for
separating the IL-2 from the butanol phase is acid precipitation.
This is done by adding the butanol phase to aqueous buffer, pH 7.5
until the organic phase is dissolved (approx. 2-3 vol buffer per
vol of organic), and then lowering the pH to about 5.5 to 7.0,
preferably 6.0 to 6.2, to cause the IL-2 to precipitate.
The next step in the process is to separate the IL-2 and any E.
coli contaminants remaining after the extraction(s) and optimally
from the solubilizing agent. Gel filtration chromatography,
RP-HPLC, or a combination of gel filtration chromatography and
RP-HPLC are used. The gel filtration chromatography is preferably
carried out in two stages that remove both pyrogenic components and
protein contaminants having molecular weights higher or lower than
IL-2. (IL-2 has a molecular weight of about 15.5K daltons.) Gels
that are capable of fractionating the solution to permit separation
of the IL-2 from these contaminants are commercially available.
Sephacryl S-200 is a preferred gel for removing the higher
molecular weight components and Sephadex G-25, G-75 or G-100 gels
are preferred for removing the low molecular weight contaminants.
The gel filtrations will typically be run in buffered solutions (pH
5.5 to 7.0) containing about 0.1% to 1.0% solubilizing agent and
about 1 to 10 mM reducing agent. The column will be sized to permit
suitable resolution of the desired components.
RP-HPLC is an alternative to gel filtration. Also, RP-HPLC is
capable of removing molecules from the solution that have molecular
weights close to IL-2 and cannot, therefore, be removed completely
by gel filtration. In addition, contaminants such as bacterial
endotoxin are also removed effectively by RP-HPLC. Therefore,
RP-HPLC may also be used as a final purification step after gel
filtration. Supports (stationary phases) that provide good
resolution of proteins may be used in the RP-HPLC. C-4, C-8, or
C-18 on 300 angstrom pore-size supports are examples of preferred
supports. The separation is carried out at an acidic pH of less
than about 2.3, usually 2.1 to 2.3, in order to keep the IL-2 in
solution. In this regard, the pH of the solution from the
solubilization (gel filtration) will preferably be adjusted to this
range. The solution is loaded into the RP-HPLC column and is
adsorbed onto the stationary phase. A gradient solvent system
comprising an organic acid such as acetic acid or trifluoroacetic
acid and organic solvent such as propanol or acetonitrile is used
to elute the IL-2 from the column. Acetic acid-propanol,
trifluoroacetic acid-propanol, and trifluoroacetic
acid-acetonitrile are preferred solvent systems. IL-2 elutes in the
acetic acid-propanol system at about 40% propanol, in the
trifluoroacetic acid-propanol system at about 50% propanol, and in
the trifluoroacetic acid-acetonitrile system at about 62%
acetonitrile. For convenience, the organic solvent content of the
elutant will usually be increased rapidly to a level somewhat below
the solvent concentration at which the IL-2 elutes followed by a
slow gradient change in the range of about 0.1% to 1.0%/min.
As soon as the IL-2 is recovered from the chromatography step, it
is lyophilized and resuspended in a neutral aqueous buffer
containing the reducing agent (to keep the IL-2 in a reduced state)
and the solubilizing agent (to keep it in solution). The IL-2 is
stable in this form and may be stored for further treatment and
formulation before being used.
An alternative and preferred procedure is to oxidize the IL-2 after
it has been separated by gel filtration and purify the oxidized
product by RP-HPLC or gel fitration followed by RP-HPLC. This
results in efficient removal of contaminants surviving the gel
filtration as well as unwanted oxidation products. A preferred
oxidation procedure is described in a commonly owned U.S. patent
application titled "Controlled Oxidation of Microbially Produced
Cysteine-Containing Proteins", U.S. Ser. No. 594,351, filed Mar.
28, 1984, now abandoned in favor of U.S. Ser. No. 661,902, filed
Oct. 17, 1984, U.S. Pat. No. 4,530,787. The relevant disclosure of
that application is incorporated herein by reference. In said
application Ser. No. 594,351 there is disclosed and claimed a
preparative process for oxidizing fully reduced cysteine-containing
microbially produced synthetic proteins, such as human IFN-.beta.
or human IL-2, in a controlled manner so that the synthetic
proteins have the same disulfide bridging as their native
counterparts. The claimed process in said application includes
oxidizing a fully reduced microbially produced synthetic protein
having an amino acid sequence substantially identical to a useful
protein which sequence includes cysteines which in the useful
protein are linked intramolecularly to form a cystine in a
controlled manner whereby said cysteines are oxidized selectively
to form said cystine with minimal over-oxidation and formation of
nonconforming cystine groups or oligomers comprising reacting the
fully reduced microbially produced synthetic protein with
o-iodosobenzoate in an aqueous medium at a pH at least about
one-half pH unit below the pKa of said cysteines and wherein the
concentration of synthetic protein in the reaction mixture is less
than about 5 mg/ml and the mole ratio of o-iodosobenzoate to
protein is at least stoichiometric, with the proviso that the
o-iodosobenzoate is in excess in the terminal portion of the
reaction. The process produces a novel cystine-containing protein,
e.g., IL-2 preparation derived from synthetic microbially produced
IL-2 having fully reduced cysteines comprising cystine-containing
IL-2 which: (i) has the same disulfide bridging as native human
IL-2; (ii) is substantially free of oligomers; and (iii) contains
less than about 15% by weight of isomers, and preferably less than
1% by weight isomers, having disulfide bridging different from
native human IL-2. RP-HPLC purification of the oxidized product may
be carried out under the conditions described above in the absence
of a reducing agent and presence of a detergent at a concentration
equal to or less than those used in the above described gel
filtration.
The purity of the IL-2 after the chromatography step(s) is at least
about 95% and usually at least about 98%. This highly pure material
contains less than about 5 ng endotoxin, usually less than about
0.01 ng endotoxin per 100,000 Units IL-2 activity.
The invention process is further described by the following
examples. These examples are not intended to limit the invention in
any manner.
EXAMPLE 1
IL-2 was recovered from E. coli K-12 strain MM294 that had been
transformed with the plasmid pLW1 (deposited at the American Type
Culture Collection on 4 Aug. 1983 under accession number 39,405) as
follows.
The E. coli were grown in a fermenter using the following growth
medium.
______________________________________ (NH.sub.4).sub.2 SO.sub.4
150 mM KH.sub.2 PO.sub.4 21.6 mM Na.sub.3 Citrate 1.5 mM
ZnSO.sub.4.7H.sub.2 O 30 mM MnSO.sub.4.H.sub.2 O 30 mM
CuSO.sub.4.5H.sub.2 O 1 mM pH adjusted to 6.50 with 2.5 N NaOH
autoclaved Sterile Additions (post autoclave) MgSO.sub.4.7H.sub.2 O
3 mM FeSO.sub.4 100 .mu.M L-tryptophan 14 mg/l Thiamine-HCl 20 mg/l
Glucose 5 g/l Tetracycline 5 mg/l Ethanol (optional) 2% Casamino
acids 2% ______________________________________
Dow Corning Antifoam B, 20% solution, glucose, 50% solution, and
KOH, 5N, were added on demand.
The pH of the fermenter was maintained at 6.8 with 5N KOH. Residual
glucose was maintained between 5-10 g/l, dissolved oxygen at 40%,
and temperature at 37.degree..+-.1.degree. C. The casamino acids
(20% stock solution) were added when the OD.sub.680 was about 10.
Harvest was made three hr after the OD.sub.680 reached about
20.
The harvested material was concentrated by hollow fiber filtration
and/or centrifugation. Twenty to forty g (wet weight) of the
concentrate were resuspended in 200 ml of 50 mM Tris, 1 mM
ethylenediaminetetraacetic acid (EDTA) (pH 8.1-8.5) (Tris/EDTA
buffer). The suspension was centrifuged at 3,000-4,000.times.g for
10 min, the supernatant was removed, and the solids were
resuspended in 200 ml Tris/EDTA buffer at 4.degree. C. The
suspension was loaded into a sonicator (Heat Systems, Model W-375)
and sonicated at 4.degree. C. for 45 min (end point=OD.sub.680
reduction of about 85%) using large probe, pulsing with 50% duty on
power setting "9". An alternative disruption technique is to pass
the suspension three times through a Manton-Gaulin homogenizer on
M-1 setting. Cellular debris was separated from the disruptate by
centrifuging at 4,500.times.g for 10 min.
The cellular debris was resuspended in 60 ml Tris/EDTA buffer at
room temperature and an equal volume of 8 M urea (Schwarz/Mann
ultrapure) in Tris/EDTA buffer was added to the suspension over
five min with rapid stirring (final urea concentration, 4 M). After
continued slow stirring for 15-30 min, the suspension was
centrifuged at 12,000.times.g for 15 min to recover extracted
cellular debris. (If a solid phase does not form, the supernatant
is withdrawn, an equal volume of Tris/EDTA buffer is added and the
mixture is recentrifuged.)
The extracted cellular debris is then resuspended in 9 ml of 50 mM
sodium phosphate (pH 6.8), 1 mM EDTA, 10 mM DTT at 20.degree. C.
One ml of 20% SDS is added to the suspension, and the suspension is
mixed vigorously for 5 min. The liquid phase is recovered from the
suspension by centrifuging at 12,000.times.g for 10 min at room
temperature. The liquid phase was then heated to 40.degree. C. for
15 min to insure that the IL-2 in the solution is fully reduced. A
sample of this crude extract was analyzed by 15% SDS-PAGE. FIG. 1
shows a densitometer scan of that analysis (product of Method 1A)
indicating the extract contained about 37% IL-2.
IL-2 was separated from the solution by gel filtration
chromatography as follows. The solution was loaded onto a 2.6
cm.times.100 cm S-200 column run in 50 mM sodium phosphate (pH
6.8), 1 mM EDTA, 1 mM DTT, 1% SDS. The column effluent was
collected in 4 ml fractions with samples of the fractions analyzed
in 15% SDS-PAGE minigels stained with Coomassie blue. The fractions
containing the fewest contaminants (minimizing contaminants at
about 35K daltons, 16-18K daltons, and 12K daltons) were pooled and
concentrated to 5-10 ml by ultrafiltration (Amicon YM5
ultrafilter). The concentrate was loaded onto a 2.6 cm.times.100 cm
G-100 column, run as above except that the SDS concentration was
0.1% rather than 1%. Fractions were analyzed by SDS-PAGE and the
purest fractions were pooled. The drawing shows a densitometer scan
of the chromatographed product. Analysis indicated the product was
98% pure and contained 0.5 ng endotoxin/ 100,000 units of IL-2
activity as measured by the limulus amebocyte lysate assay
(Associates of Cape Cod, Inc., Woods Hole, MA). The N-terminal
amino acid sequence of this IL-2 is the same as the native human
molecule except that the initial N-terminal alanine is missing.
EXAMPLE 2
The procedure of Example 1 was repeated using 2% sarcosyl instead
of 2% SDS as a solubilizing agent and using sarcosyl in place of
SDS in the chromatography columns. FIG. 1 shows the densitometer
scan for this crude extract using sarcosyl as a solubilizing agent
(crude extract of Method 1B). As indicated, the use of sarcosyl in
place of SDS gave improved purity (58% vs 37%) at similar IL-2
yield (50% vs 60%).
EXAMPLE 3
The procedure of Example 1 was repeated through the steps preceding
urea extraction and was then solubilized and clarified as
described.
The IL-2 was separated from the solution by RP-HPLC as follows. The
solution was diluted 10-fold in 0.1% trifluoroacetic acid (TFA) and
was applied to a 4.6 mm I.D..times.5 cm L. Brownlee Aquapore RP-300
column equilibrated in 0.1% TFA. The IL-2 was eluted with a
gradient of 30%-60% acetonitrile containing 0.1% TFA over 45 min.
The yield of IL-2 activity following HPLC was 80-100%. FIG. 2 shows
a silver-stained SDS-PAGE analysis of this product.
EXAMPLE 4
The procedure of Example 1 was repeated through the steps preceding
get filtration chromatography. The soluble, clarified, reduced
material was subjected to G-100 chromatography in 0.1% SDS as
described in Example 1. The pooled peak fractions of IL-2 were
further purified by RP-HPLC as described in Example 3. The
resulting purified, reduced IL-2 was oxidized and subjected to
RP-HPLC as described in Example 3.
EXAMPLE 5
The procedure of Example 1 was repeated through the steps preceding
the G-100 column. The procedure for Example 3 was repeated using a
solvent system of propanol in 1 M acetic acid. The IL-2 was eluted
with a gradient of 35%-60% propanol over 200 min. Column dimensions
were either 10 mm ID.times.30 cm L or 48 mm ID.times.50 cm L, and
the column was packed with a bonded phase wide-pore silica gel. The
bonded phase wide-pore silica used was Vydac TP214. The purity and
yield of product was comparable to that of Example 3.
EXAMPLE 6
The procedure of Example 3 was repeated using a solvent system of
propanol in 0.1% TFA. The IL-2 was eluted with a gradient of
35%-60% propanol over 120 min. The column and support materials
were the same as in Example 5. The purity and yield of product were
comparable to that of Example 3.
EXAMPLE 7
The procedure of Example 1 was repeated except that the E.
coli-produced IL-2 was one designated des-Ala Ser.sub.125 IL-2. The
amino acid sequence of this IL-2 is different from that of the
native molecule in that the cysteine at position 125 has been
changed to serine and the initial N-terminal alanine residue is
missing. The E. coli that produce this IL-2 were made by the
techniques described in said U.S. Ser. No. 4,518,584. Strains of
des-Ala Ser.sub.125 IL-2-producing E. coli were deposited in the
American Type Culture Collection on Sept. 26, 1983 under accession
number 39,452 and on Mar. 6, 1984 under accession number
39,626.
EXAMPLE 8
The procedure of Example 1 was repeated except that the IL-2 was
recovered from E. coli K-12 strain that had been transformed with
the plasmid pLW55 (deposited in the American Type Culture
Collection on Nov. 18, 1983 under accession number 39,516). The
amino acid sequence of this molecule is different from that of the
native molecule in that it has an N-terminal methionine and the
cysteine at position 125 has been changed to serine.
EXAMPLE 9
Des-Ala Ser.sub.125 IL-2-producing E. coli were grown, the cells
disrupted and the cellular debris recovered from the disruptate
using the general procedures of Example 1. The cellular debris was
suspended in 50 mM Tris, 1 mM EDTA pH 8.5 buffer at a ratio of
about 1:4.5 (w/v). DTT was added to a final concentration of 25 mM.
8 M urea in the same buffer was slowly added with stirring to a
final concentration of 4 M and then allowed to mix at room
temperature for 30 min. After 30 min, the insoluble material
remaining was centrifuged. The resulting paste was resuspended in
50 mM sodium phosphate buffer, 1 mM EDTA pH 7.0. The suspension was
then solubilized by addition of solid SDS to a final concentration
of 5% w/v.
The 5% SDS solution was diluted to 2% SDS with 0.1 M Na.sub.2
PO.sub.4, pH 8.0. The protein concentration was determined, the pH
was adjusted to 8.5, and DTT to 50 mM and EDTA to 2 mM were added.
The mixture was heated to 40.degree. C. under N.sub.2 to reduce the
IL-2. The mixture was then cooled and the pH was adjusted to
5.0.
The solution was then extracted at a 1:1 ratio (v/v) with 2-butanol
containing 1 mM DTT at room temperature. Residence time was 2-2.5
min. The extraction was carried out in a liquid-liquid phase
separator using a flow rate of 200 ml/min. The organic extract was
separated and its pH was adjusted to 8.0 with NaOH. The extract was
then added slowly to 0.1% SDS in 10 mM Na.sub.2 PO.sub.4, 2 mM DTT,
pH 6 and stirred for 15-20 min. The resulting precipitate was
separated and the resulting paste was resuspended in 5% SDS in PBS.
The solution was clarified by centrifugation and reduced as above.
Following reduction the solution was adjusted to pH 5.5 with acetic
acid. The solution was purified by gel filtration using S-200 and
G-25 columns. The resulting purified, reduced IL-2 was oxidized,
and the oxidized product was purified by G-25 chromatography
followed by RP-HPLC as in Example 3. The resulting purified
recombinant IL-2 product has an IL-2 content greater than about 95%
as determined by reducing SDS-PAGE analysis, an endotoxin content
of less than about 0.1 nanograms/mg of IL-2, and it is
substantially free of pyrogens as determined by the U.S.P. rabbit
pyrogen test at a dosage of 3.3.times.10.sup.5 U/kg. As previously
indicated, the endotoxin content is less than about 5 nanograms,
and preferably less than 0.01 nanograms endotoxin per 100,000 units
IL-2 activity. Typically, the purified recombinant IL-2 products
purified by the process of the invention have an IL-2 content
greater than 98% as determined by reducing SDS-PAGE or RP-HPLC, as
shown in FIG. 3 in addition to being substantially free of
endotoxins and pyrogens as indicated above.
A variation of the process described in Example 9, such as might be
used to produce IL-2 on a larger scale, is shown in FIG. 4. The
process shown in FIG. 4 differs from that described in Example 9 as
regards (1) minor changes in the buffers, (2) use of an acetic
acid-propanol (Example 5) solvent system in the RP-HPLC, and (3)
the inclusion of post-oxidation dilution/diafiltration S-200 gel
filtration, and ultrafiltration steps. The process as shown in FIG.
4 may be modified with various refinements, for example, following
the second S-200 column pass, in 1% SDS, the IL-2 solution is
diluted 1:10 to give a 0.1% SDS concentration and then diafiltered
against 10 mM phosphate buffer at a pH of 7.5 and 5 ppm SDS. The
solution is then concentrated as required for appropriate use
dosage.
Modifications of the above described modes for carrying out the
invention that are obvious to those of skill in the fields of
biochemistry, biochemical engineering, and related arts are
intended to be within the scope of the following claims.
* * * * *